Polyomavirus diagnostic reagents

ABSTRACT

The present invention relates to HLA-A*02-restricted cellular epitopes within the VP1 polypeptide of human polyomaviruses, which are useful as diagnostic reagents for virus infection. Preferred peptides correspond to amino acids residues 107-116, 108-116 and 44-52 of BKV VP1, and are processed in vivo in natural infection with BKV. Effector T cell populations stimulated by the peptides represent functional CTLs as assessed by cytotoxicity and cytokine production, and are reactive against cells presenting both the BKV peptides above and the JC virus homolog sequences.

This application is a divisional of prior co-pending U.S. applicationSer. No. 11/491,542, filed Jul. 24, 2006, which claims the benefit ofprior co-pending U.S. Provisional Application Ser. No. 60/701,484, filedJul. 22, 2005. Both of these applications are hereby incorporated byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support in the form of grantsno. 1R21CA104261-01 from the National Cancer Institute of the UnitedStates Department of Health and Human Services, National Institutes ofHealth. The United States government may have certain rights in theinvention.

BACKGROUND OF THE INVENTION

1. Technical Field

This invention relates to the field of medical sciences, in particularthe field of immunology and viral immunity. Specifically, the inventionrelates to cellular epitopes of the VP1 polypeptide of polyomaviruses,for example BK virus, JC virus and SV40.

2. Description of the Background Art

Polyomavirus hominis 1, known as BK virus (BKV), is a ubiquitous humanpolyomavirus that causes asymptomatic primary infection and resides,latent, in several body sites, notably the kidney and genitourinarytract. See Stolt et al., J. Gen. Virol. 84:1499-1504, 2003. BKV, therelated polyomavirus hominis 2 (also known as JC virus or JCV) andsimian vacuolating virus 40 (SV40) are closely related species of thegenus polyomavirus. The BKV genome is about 75% homologous overall toJCV and about 70% homologous overall to SV40.

Primary BKV infection during childhood is presumed to occur via therespiratory tract. Following infection, hematologous dissemination ispostulated to occur. Persistence is preferentially within thegenitourinary tract (renal epithelial cells and the lower genitourinarytract (bladder, ureters)). Chesters et al., J. Infect. Dis. 147:676-684,1983. BKV reactivation often occurs in immunocompromised individualssuch as hematopoietic stem cell transplant and kidney transplantrecipients, causing clinical disease states which include hemorrhagiccystitis, ureteric stenosis and polyomavirus-associated nephropathy(PVAN, also referred to as BK virus-associated nephropathy (BKVN)). Theincidence of polyomavirus-associated nephropathy, variously reported asbetween 1% and 8% of kidney transplant patients, has increased in recentyears, concomitant with the use of newer and more potentimmunosuppressive agents in transplant patients, suggesting that immuneresponses to BKV are important in control of the virus. Hirsch andSteiger, Lancet 3:611-623, 2003.

Polyomavirus associated nephropathy now is recognized as an importantcause of allograft dysfunction in kidney transplant recipients. Binet etal., Transplantation 678:918-922, 1999; Hurault et al., Transplant Proc.32:2760-2761, 2000; Randawa et al., Transplantation 67:103-109, 1999.With immunosuppression, viral reactivation can occur, presumablyprimarily from the lower genitourinary tract, and is detected as virusshed in the urine (viruria). With continued immunosuppression and otherlocal injury within the kidney, such as rejection or calcineurininhibitor toxicity, polyomavirus reactivation also can occur in thekidney itself, leading to viral replication, direct injury to theinfected tubular epithelial cells and indirect injury manifested byinflammation and nephritis, ultimately leading to rejection of thekidney in some cases. In kidney transplant recipients, BKV reactivationis particularly associated with interstitial nephritis/nephropathy andureteric stenosis. Nickeleit et al., J. Am. Soc. Nephrol. 10:1080-1089,1999; Nickeleit et al., Nephrol. Dial. Transplant. 15:324-332, 2000.

Other than the antiviral agent, cidofovir, for treatment of PVAN, noother antiviral therapies are available for polyomavirus. Kadambi etal., Am. J. Transplant. 3:186-191, 2003; Scantlebury et al., Graft5(supp):S82-S87, 2002; Vats et al., Transplantation 75:105-112, 2003.Cidofovir requires intravenous administration and is associated withconsiderable nephrotoxicity itself, particularly in patients withpre-existing nephrotoxicity. Based on a consensus opinion that PVAN mayrepresent a state of relative over-immunosuppression, the currentapproach to managing patients with PVAN is reduction ofimmunosuppression. Even using this approach, however, up to 30-50% ofpatients with PVAN develop progressive polyomavirus infection anddeterioration of kidney function, ultimately resulting in loss of thekidney allograft. Hirsch and Steiger, Lancet 3:611-623, 2004; Nickeleitet al., Nephrol. Dial. Transplant. 15:324-332, 2000; Randhawa et al.,Transplantation 67:103-109, 1999. Given the limited treatment options,there is an urgent need for alternative approaches to protect againstBKV reactivation and disease.

JCV also is prevalent worldwide, with about 80% of adults showingserological evidence of JCV infection. Stolt et al., J. Gen. Virol.84:1499-1504, 2003. Most individuals are infected in childhood, withoutshowing any symptoms. The virus remains latent in the lymphocytes,urogenital tract and brain and can reactivate in the immunocompromised,causing disease syndromes. JCV is the causative agent of progressivemultifocal leukoencephalopathy (PML), a fatal degenerative diseaseaffecting brain oligodendroglial cells seen in immunosuppressed AIDS,cancer and organ transplant recipient patients. JCV also is associatedwith hemorrhagic cystitis and nephritis in kidney transplant recipients.

SV40 is a closely related polyomavirus of simians which also widelyinfects humans and has been associated with some tumor types. This virushas a high degree of serological cross-reactivity with both BKV and JCVantigens. SV40 is believed to spread through a respiratory or fomiteroute and to be established as a human pathological agent. Its presumedsite of persistence also is the kidney, and other tissues that give riseto SV40-associated tumors (e.g., mesothelioma, lymphoma, osteosarcoma,and certain brain tumors).

The capsid of polyomavirus is largely made up of VP1, VP2 and VP3, witheach virion containing 360 copies of VP1. Two cellular epitopes of JCVVP1 have been identified, including the epitope sequences, ILMWEAVTL(JCV VP1_(p100-108); SEQ ID NO:3; referred to herein as “JC100”) andSITEVECFL (JCV VP1p36-44; SEQ ID NO:6; referred to herein as “JC36”). DuPasquier et al., J. Neurovirol. 7:318-322, 2001; Du Pasquier et al., J.Virol. 77:11918-11926, 2003; Du Pasquier et al., Brain 127(9):1970-1978,2004; Du Pasquier et al., J. Virol. 78:10206-10, 2004; Koralnik et al.,J. Immunol. 168:499-504, 2002; Koralnik et al., J. Virol. 75:3483-3487,2001. CTL recognizing these epitopes have been associated with controlof the virus; patients suffering from the JCV syndrome, progressivemultifocal leukoencephalopathy, demonstrate a prolonged survival whenthey possess CTL responses to the JC100 epitope. Du Pasquier et al.,Brain 127(9):1970-1978, 2004; Koralnik et al., J. Immunol. 168:499-504,2002. BKV-specific cells also have been detected in samples from renaltransplant patients. Comoli et al., Transplantation 78:1229-1232, 2004.These prior studies, which used BKV-infected cell lysates as antigens,did not identify any specific antigens or epitopes, however.BKV-specific T-cell lines have been developed from healthy seropositiveindividuals and kidney transplant recipients. Comoli et al.,Transplantation 78:1229-1232, 2004; Comoli et al., J. Am. Soc. Nephrol.14:3197-3204, 2003; Drummond et al., J. Med. Virol. 17:237-247, 1985;Drummond et al., J. Med. Virol. 23:331-344, 1987.

Several previous studies have shown that both JCV and BKV are common inmost adult populations, but that JCV is less prevalent than BKV. Knowleset al., J. Med. Virol. 71:115-123, 2003; Padgett et al., J. Infect. Dis.127:467-470, 1973; Taguchi et al., Microbiol. Immunol. 26:1057-1064,1982. Knowles and colleagues reported 81% seropositivity for BKV and 35%for JCV in a survey of 2,435 sera from 1991. Knowles et al., J. Med.Virol. 71:115-123, 2003. These types of studies are complicated bysubstantial serological crossreactivities between antibodies to thesetwo human polyomaviruses. Viscidi et al., Clin. Diagn. Lab Immunol.10:278-285, 2003. However, antibody adsorption studies show that someindividuals experience infection by both JCV and BKV. This is furthersupported by PCR studies of polyomavirus shedding in urine that indicateco-infection in a minority of patients. Bendiksen et al., J. Gen. Virol.81:2625-2633, 2000; Hamilton et al., J. Clin. Microbiol. 38:105-109,2000; Priftakis et al., J. Clin. Microbiol. 38:406-407, 2000; Shah etal., J. Infect. Dis. 176:1618-1621, 1997.

Knowledge of specific MHC—I restricted epitopes within BK virus antigensallows one to track virus-specific CTLs in at-risk patients and producecompositions to modify immunity to BK and related viruses such as JCVand SV40. Peptides identified as immunoreactive are useful for vaccinesas well as diagnostic reagents. Vaccine peptides from viral proteins maybe used for enhancing the immune system of mammals with respect to thevirus in both seropositive and seronegative individuals. However, nosuch specific information concerning BKV epitopes has been available.There is a need in the art for compositions and methods which can beused to identify and diagnose polyomavirus-related conditions inpatients and patient-derived samples, and to modify the immune responseof patients in need, such as immunosuppressed patients, or any person atrisk for polyomavirus infection or reactivation.

SUMMARY OF THE INVENTION

Accordingly, the invention generally relates to a viral epitope usefulfor diagnosis and treatment of human polyomavirus. In one embodiment,the invention provides the isolated peptides LLMWEAVTV (SEQ ID NO:1),AITEVECFL (SEQ ID NO:5) and NLLMWEAVTV (SEQ ID NO:2). In otherembodiments, the invention provides vaccine compositions againstpolyomavirus that comprise these peptides. Such vaccines are useful forpolyomavirus prophylaxis and treatment, including BKV, JCV and SV40.Vaccines according to embodiments of the invention optionally alsocomprise an adjuvant, for example a DNA adjuvant and/or a T helperepitope such as PADRE. In addition, vaccines of the invention includeantigen presenting cells that present the peptide LLMWEAVTV (SEQ IDNO:1), AITEVECFL (SEQ ID NO:5) or NLLMWEAVTV (SEQ ID NO:2) in oneembodiment, or a viral vaccine that encodes LLMWEAVTV (SEQ ID NO:1),AITEVECFL (SEQ ID NO:5) or NLLMWEAVTV (SEQ ID NO:2).

In further embodiments, the invention provides a polyomavirus diagnosticreagent which comprises the peptide LLMWEAVTV (SEQ ID NO:1), AITEVECFL(SEQ ID NO:5) or NLLMWEAVTV (SEQ ID NO:2), for example a tetramerreagent.

In yet a further embodiment, the invention provides a method ofexpanding polyomavirus-specific cytotoxic T lymphocytes in a populationof T lymphocytes which comprises contacting the population of Tlymphocytes with antigen presenting cells that present the peptideLLMWEAVTV (SEQ ID NO:1), AITEVECFL (SEQ ID NO:5) or NLLMWEAVTV (SEQ IDNO:2) and such expanded polyomavirus-specific cytotoxic T lymphocytes.

In yet a further embodiment, the invention provides a method ofmodulating the immune response of a patient to a polyomavirus orimmunotherapy for treatment of a polyomavirus-infected patient whichcomprises administering to the patient any of the vaccines discussedabove and/or the polyomavirus-specific cytotoxic T lymphocytes discussedabove.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides results of a T2 stabilization/HLA-A02 binding assaycomparing six BKV VP1 peptides and two control peptides. A: no peptide;B: BK26 (KLLIKGGVEV; SEQ ID NO:8); C: BK27a (LLIKGGVEVL; SEQ ID NO:10);D: BK27b (LLIKGGVEV; SEQ ID NO: 9); E: BK108 (LLMWEAVTV; SEQ ID NO:1);F: BK107 (NLLMWEAVTV; SEQ ID NO:2); G: BK109 (LMWEAVTVQT; SEQ ID NO:11);H: control peptide (ILKEPVNGV; corresponding to part of the HIV POL openreading frame; SEQ ID NO:12); I: control peptide (QIKVRVDMV;corresponding to a HLA-B*08-restricted epitope in the HCMV majorimmediate-early gene; SEQ ID NO:13).

FIG. 2 shows results of a cytotoxicity assay of the cells of FIG. 3using JA2 cells loaded with BK108 or control HIV peptide. Diamond: BK108peptide; square: control peptide.

FIG. 3 shows results of ICC assays on splenocytes from HHD-II miceimmunized with BK108 peptide, the T helper peptide, PADRE, and CpG DNAadjuvant.

FIG. 4 shows results of a cytotoxicity assay of splenocytes from HHD-IImice immunized with rMVA expressing BKV VP1. These splenocytes werestimulated in vitro with BK108, and the cytotoxicity assays used JA2cells loaded with BK108 or control HIV peptide as targets. Diamond:BK108 peptide; triangle: BK108 peptide: square: control peptide.

FIG. 5 shows ICC assay results of the cells described for FIG. 4.

FIG. 6 shows results of specific cytotoxicity of transgenic splenocytesexpressing BKV VP1 using A2-Jurkat cells pulsed with the indicatedpeptides.

FIG. 7 shows results of an ICC assay of immunized transgenic micesplenocytes (rMVA-BKV VP1) stimulated in vitro with the peptides BK108(7A), JC100 (7B) and control peptide (7C).

FIG. 8 shows the results of intracellular cytokine (ICC) assays forIFN-γ production on peptide stimulation of transgenic splenocytesexpressing BKV VP1 with the indicated peptides.

FIG. 9 shows results of screening PBMC from healthy donors and kidneytransplant recipients for CTL recognizing the BK108 epitope. All plotsare gated on lymphocytes by forward vs. side scatter.

FIG. 10 shows the functionality of BK108 tetramer-binding cells fromnormal donor #07, labeled with CyChrome™-conjugated antibody to CD8.

FIG. 11 shows the functionality of BK108 tetramer-binding cells fromnormal donor #07, labeled with PE-conjugated antibody to IFN-γ.

FIG. 12 shows fluorescence-activated cell sorting results for PBMC andpeptide-stimulated cell cultures from a kidney transplant recipient. Thecells were labeled with either BK108tet-APC (12A, 12B and 12C) orJC100tet-PE (12D, 12E and 12F) and anti-CD8 FITC.

FIG. 13 shows fluorescence-activated cell sorting results for PBMC andpeptide-stimulated cell cultures from a kidney transplant recipient. Thecells were labeled with both BK108tet-APC and JC100tet-PE.

FIG. 14 shows representative results of a cytotoxicity assay for immunesplenocytes against Jurkat A2 targets that had been pulsed with BK44peptide, JC36 peptide or an irrelevant HIV peptide.

FIG. 15 provides results of an intracellular cytokine assay performed onthe cells analyzed in FIG. 14.

FIG. 16 provides data from flow analyses of an aliquot of PBMC from anormal donor, stimulated in culture with BK44 peptide in the presence ofrIL2 and labeled with BK44tet-APC (FIG. 16A) or JC36tet-PE (FIG. 16B).

FIG. 17 provides data from flow analyses of an aliquot of PBMC from anormal donor, stimulated in culture with BK44 peptide in the presence ofrIL2 and labeled with both BK44tet-APC and JC36tet-PE.

FIG. 18 is a bar graph summarizing the data from analyses of the typeillustrated in FIGS. 16 and 17 on PBMC from 8 healthy normal HLA-A*02donors.

FIG. 19 provides flow cytometry staining results after in vitrostimulation with BK108 peptide, using BK108 tetramer reagent.

FIG. 20 provides flow cytometry staining results after in vitrostimulation with JC100 peptide, using JC100 tetramer reagent.

FIG. 21 provides flow cytometry double-staining results after in vitrostimulation with BK108 peptide, using both BK108 and JC100 tetramerreagents.

FIG. 22 provides flow cytometry double-staining results after in vitrostimulation with JC100 peptide, using both BK108 and JC100 tetramerreagent.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The BK virus sequences LLMWEAVTV (SEQ ID NO:1; referred to herein as“BK108”) and AITEVECFL (SEQ ID NO:5; referred to herein as “BK44”) havebeen identified as immunodominant epitopes within the VP1 polypeptide ofBK virus. These epitopes are restricted by HLA-A2 and are recognized bycytotoxic T lymphocytes (CTL) both in transgenic mice immunized withrecombinant vaccinia virus and in humans naturally infected with BKV. Inaddition, CTL specific for these BKV epitopes can cross-recognizeantigen presenting cells displaying the JC virus and SV40 homologs ofthe epitope. See Table I below for related sequences.

TABLE I Polyomavirus Epitope Peptides. SEQ ID Virus Name Sequence NO BKVBK108 LLMWEAVTV 1 BKV BK107 NLLMWEAVTV 2 JCV JC100 ILMWEAVTL 3 SV40SV108 ILMWEAVTV 4 BKV BK44 AITEVECFL 5 JCV JC36 SITEVECFL 6 SV40 SV44SFTEVECFL 7

BK virus is a clinically important polyomavirus for which no antigenicepitopes were previously known. The BK108 and BK44 peptides are nineamino acids in length, and therefore correspond to the presumptivelength of a minimal antigenic sequence. Using the BK108 and BK44sequences, one can prepare specific probes, reagents and cells such as,for example, peptides containing the sequence or tetramer reagentsfolded with the sequence to track CTL specific for polyomavirus in humanpatients, for diagnostic or monitoring purposes, or antigen presentingcells or peptide vaccines for treatment and prophylaxis ofpolyomavirus-related disease. Tetramer reagents are described inBieganowska et al., J. Immunol. 162:1765-1771, 1999 and Lebowitz et al.,Cell Immunol. 192:175-184, 1999, the disclosures of which are herebyincorporated by reference.

CTL that recognize BK108 or BK44 can be protective against BKV or otherpolyomaviruses, for example the human JC virus and the simian virusSV40, both of which can infect humans and cause disease. ClinicalBKV-related disease includes, but is not limited to,polyomavirus-associated nephropathy, hemorrhagic cystitis and prostateneoplasm. Many of these conditions have been associated withimmunosuppression or related conditions. Patient populations who maytake particular advantage of the present invention include, but are notlimited to, kidney transplant patients, hematopoietic stem celltransplant patients, AIDS patients, prostate cancer patients andpolyomavirus-negative persons. Any solid organ transplant patient orimmunosuppressed person also may advantageously be treated withcompounds comprising BK108 and/or BK44. BK108 and BK44 therefore can beuseful as vaccines, for prophylaxis or treatment of infection with thesepolyomaviruses. Passive or active immunotherapy using cells presentingthe BK108 and/or BK44 peptides can be useful in any patient infectedwith or at risk for infection by polyomaviruses. Thus, BK108 and BK44form the basis for a peptide-based vaccine against polyomavirus. Thepeptides also are useful to expand polyomavirus-specific CTL foradministration as adoptive T cell immunotherapy against polyomavirus.

In the studies reported here, immunization of transgenic mice that modelthe human immune system with rMVA expressing BKV VP1 produced CTL thatrecognize BK108 (LLMWEAVTV; SEQ ID NO:1) and cross-recognize the JCV VP1homolog sequence JC100 (ILMWEAVTL; SEQ ID NO:3), as well as CTL thatrecognize BK44 (AITEVECFL; SEQ ID NO:5) and cross-recognize JC36(SITEVECFL; SEQ ID NO:6). In addition, healthy BKV-seropositive HLA-A2individuals and a kidney transplant recipient patient were shown toharbor low frequencies of CTL precursors that can be expanded bystimulation with BK108, JC100 or BK44 peptide into functional CTL thatrecognize both the relevant epitope and the BKV or JCV homolog sequence.The significantly higher levels of BKV/JCV VP1-specific CD8⁺ T cellsseen in one of the two kidney transplant recipients tested, compared tothe ten normal donors examined, suggests that the documented BKVreactivation in this individual drove expansion of CTL precursorsspecifically recognizing this epitope.

Du Pasquier et al. identified the JC100 and JC36 epitope sequences usinga computer-based epitope prediction method. Du Pasquier et al., Brain127(9):1970-1978, 2004; Du Pasquier et al., J. Virol. 78:10206-10210,2004; Koralnik et al., J. Immunol. 168:499-504, 2002; see also Koralniket al., J. Immunol. 168:499-504, 2002. Using JC100 and JC36 epitopepeptides and tetramer reagents incorporating the peptides, thoseinvestigators identified CTL recognizing these epitopes in HLA-A*02 HIV⁺PML survivors and in healthy individuals for whom the BKV serostatus wasnot reported. Correlation of clinical PML status and the presence of CTLrecognizing these epitopes demonstrated an association between thesecells and early control of PML. Du Pasquier et al., Brain127(9):1970-1978, 2004; Koralnik et al., J. Immunol. 168:499-504, 2002.However, the data presented here show that the JCV VP1p100 and JCVVP1p36 epitope peptides and tetramer reagents based on these peptidesare not JCV-specific, since they cross-react with cells elicited inresponse to and recognizing the BKV homolog peptide. Cellular responsesrecognizing the JC100 or JC36 epitopes in previous studies therefore mayhave been the result of JCV infection, BKV infection or infection withboth viruses.

Given that the previously studied cellular immune responses are reportedto protect against PML (Du Pasquier et al., J. Neurovirol. 7:318-322,2001; Du Pasquier et al., Brain 127(9):1970-1978, 2004; Koralnik et al.,J. Immunol. 168:499-504, 2002), prior BKV infection may cross-protectagainst JCV disease. The studies reported here suggest that at-riskpopulations for BKV and/or JCV disease, and for SV40, can be monitoredusing diagnostic reagents based on BK108 (SEQ ID NO:1) or on BK44 (SEQID NO:5). In addition, immuno-interventive therapies based on thesepeptides can be targeted against the polyomaviruses BKV, JCV and SV40.

Those of skill in the art are fully able to devise schemes andcompositions for vaccination and/or immune system modification using thepeptide of this invention, and these variations are contemplated for usewith the present invention. The following description, therefore, isintended to provide guidance and not to be limiting in any way.

The peptide of the invention may be formulated as vaccines according toany suitable method. Naked peptide or lipidated peptide may beformulated in any pharmaceutically acceptable carrier known in the art.Preferred peptide vaccine compositions also comprise an adjuvant. DNAadjuvants are preferred for human use. The peptides may be formulated asfusions with other immunogenic peptides from the same or a differentpathologic entity. Peptides may be synthesized as fusions of the BK108or BK44 peptides with one or more T-helper epitope such as PADRE orcertain known tetanus peptides. Spacer peptides also may comprise partof these fusions.

The peptides may be formulated for any suitable mode of administration,however subcutaneous, intradermal, intramuscular, mucosal (e.g., rectal,nasal, vaginal, etc.), intraperitoneal, transdermal or inhalant modes ofadministration are preferred. Those of skill in the art ofpharmaceutical formulation are well aware of appropriate and suitablecarriers, diluents, excipients and other ingredients which may be usedto create formulations for these modes of administration, and any ofthese compounds and formulations are contemplated for use with theinvention. For human administration of a peptide or peptide fusioncomposition, a first immunization of about 10 mg to about 10,000 mg orpreferably about 25 to about 2500 mg peptide is appropriate, followed byone, two or more booster immunizations at intervals (about 2-6 weeks),if desired.

The invention also includes DNA vaccines that encode the BK108 and/orBK44 peptides. Such DNA vaccines and methods for their formulation areknown in the art. Generally, such vaccines are administered topreviously infected or uninfected patients, or in vitro to T cells, inthe form of a polynucleotide. A suitable gene-transfer vector such as aplasmid or engineered virus vector (for example MVA) is prepared tocontain and express DNA that encodes the peptide or a peptide fusion,under the control of one or more appropriate expression regulatorysequences. T cells transfected in vitro with the DNA based vaccine maybe administered to persons as well. For DNA immunizations, the patientpreferably is injected intramuscularly with about 0.1-5 mgendotoxin-free DNA diluted in sterile saline or any other suitablepharmaceutical carrier, according to known methods.

Cellular vaccines and antigen presenting cells incorporating the BK108and/or BK44 peptides also form part of the invention. Such cells andcellular vaccines are antigen-presenting cells that have been treated invitro to cause them to present the peptide, for example, by in vitroincubation with (50 μM) BK108 and/or BK44 peptide for about 1-4 hours,followed by washing. Alternatively, the cells may be infected with atransfer virus vector containing DNA that encodes the peptide(s). TheDNA construct for DNA vaccines may consist of a mammalian expressionvector such as pVAX (Invitrogen™) in which the DNA sequence of each ofthe peptides of interest are inserted in the multicloning site,separated by spacers. For production of cellular vaccines, the describedDNA construct may be electroporated into appropriate cells such asautologous dendritic cells.

The person of skill is readily able to determine patients who willbenefit from vaccination or immune system modification with respect topolyomaviruses. In general, any person at risk of infection(prophylactic vaccine), such as children, or any person at risk ofpolyomavirus reactivation and disease (treatment vaccine), such as solidorgan or other transplant recipient patients or donors, AIDS patients,cancer patients or any immunosuppressed person is a suitable candidatefor the compositions and methods of this invention.

An additional aspect of the invention relates to diagnostic reagents fordetection of polyomavirus infections. The BK108 and BK44 peptides canstimulate CTL directly in vitro and therefore can be used in an assay todetermine the degree of immunostimulation being caused by polyomavirusessuch as BKV, JCV and SV40. The peptides also can be used to diagnoseindividuals who are infected with polyomavirus. For use as a diagnosticreagent, for example for the detection of active versus quiescent BKV orother polyomavirus infection, the BK108 and/or BK44 peptides (orantigen-presenting cells presenting the peptide(s)) are contacted invitro with a patient sample containing T cells according to the methodsdescribed in the Examples. Expansion of T cell clones recognizing thepeptide from the patient sample indicates the presence of BK108- orBK44-reactive CTL and therefore polyomavirus infection. Bissinger etal., Exp. Hematol. 30:1178-1184, 2002, the disclosures of which arehereby incorporated by reference, have described the use of anintra-cellular cytokine (ICC) assay to expand HCMV-specific CTL withIL-2 and feeder cell stimulation using an HCMV epitope peptides. Usingthis method, not only can the ICC assay determine whether the subject isreactive to a particular peptide, but cells reacting to the peptide canbe isolated and expanded to be used for adoptive immunotherapy.

Alternatively, tetramer reagents and the like, which are known in theart (see U.S. Pat. No. 5,734,023, the disclosures of which are herebyincorporated by reference) may be constructed from the peptides of theinvention to detect T cells that recognize BK108 or BK44. See Lacey etal. Transplantation 74:722-732, 2002, the disclosures of which arehereby incorporated by reference, and Example 4 for appropriate methods.Tetramer reagent-positive cells may be transferred into the recipient inwhom expansion is desired, to protect against polyomavirus-relateddisease.

EXAMPLES Example 1 Identification of HLA-A*02-Restricted Epitopes of BKVVP1

Sequences with the motif characteristics of T-cell epitopes wereidentified within the open reading frames encoding the BKV VP1 majorcapsid polypeptide using computer-based algorithms that predict 9- or10-mer amino acid sequences likely to be generated by proteasomalcleavage and to bind to HLA-A*02. The SYFPEITHI™, BIMAS™, SVMHC™ andFRAGPREDICT™ algorithms were used to select the panel of 6 candidateepitopes shown in Table II, below.

TABLE II Predicted HLA-A2 Restricted Epitopes from BKV VP1. SEQ PeptideVP1 ID Name Position Sequence NO: BK26  26-35 KLLIKGGVEV 8 BK27a  27-36LLIKGGVEVL 10 BK27b  27-35 LLIKGGVEV 9 BK108 108-116 LLMWEAVTV 1 BK107107-116 NLLMWEAVTV 2 BK109 109-118 LMWEAVTVQT 11

Peptides were synthesized using standard FMOC protocols using a SymphonyQuartet™ peptide synthesizer and purified to greater than 95% purity byHPLC. The identity of the peptides was confirmed by MALDI TOF massspectrometric analysis using a Kompact Probe™ mass spectrometer. Thesepeptides then were tested for their ability to bind HLA-A*02 andstabilize its expression on the surface of TAP-deficient T2 cells.Levels of surface HLA-A2 were measured by staining peptide-pulsed T2cells with a fluorescent-labeled antibody to HLA-A2 followed by flowcytometric analysis. Five of the six VP1 peptides clearly showedpositive HLA-A2 binding as measured by an increase in relativefluorescence intensity on the surface of the cells.

See FIG. 1 for results of a representative cell-binding assay. Thedegree of binding to T2 cells in culture is indicated by the degree ofdisplacement (to the right) of the overlaid histograms on the X-axis ofthe Figure, which indicates increased fluorescence, corresponding tohigher levels of stabilized peptide-HLA complex on the surface of the T2cells. Peptides BK108, BK27b and BK107 demonstrated the highest bindingfunction, comparable to a positive control HLA-A2-binding peptidecorresponding to a well-defined immunodominant epitope from the humancytomegalovirus pp65 polypeptide (NLVPMVATV; SEQ ID NO:14).

Example 2 In Vivo Immunogenicity Testing in Transgenic Mice

Peptides BK26, BK27b, BK108, and BK107 were used to immunize HHD-IItransgenic mice, with a CpG-rich oligodeoxynucleotide and PADRE T-helperpeptide in incomplete Freund's adjuvant. These HHD-II mice have ahumanized immune system and are well-recognized to predict immuneresponses in humans. The mice express a transgenic monochainhistocompatability class I molecule in which the C terminus of the humanβ2m is covalently linked to the N terminus of a chimeric heavy chain(HLA-A*0201-α1, -α2, H-2D^(b)-α3-transmembrane, and intracytoplasmicdomains). See Firat et al., Eur. J. Immunol. 29:3112-3121, 1999, thedisclosures of which are hereby incorporated by reference. Fourteen daysafter immunization, the mice were sacrificed, the spleen removed, andparallel in vitro stimulation cultures were set up using each of thefour peptides above.

In vitro stimulation of PBMC was performed as follows. CryopreservedPBMC were cultured in 24-well tissue culture plates at a density of 3.5million cells/mL in RPMI 10 containing 1 μg/mL of either BK108 (SEQ IDNO:1) or JC100 (SEQ ID NO:3) at 37° C. in a CO₂-gassed incubator. After3 days of culture, recombinant human IL-2 (rIL-2) was added to 30units/mL. Every two days thereafter, 50% of the culture medium wasremoved and replaced by fresh medium containing rIL-2. Incubation wascontinued for 11-14 days before flow analysis.

The stimulated cultures were tested for cytotoxicity againstantigen-presenting Jurkat cells loaded with the peptide with which theywere stimulated. Flow cytometric intracellular cytokine (ICC) assaysalso were used to test the stimulated cultures for the presence of CD8⁺T cells that produce IFN-γ in response to stimulation by the cognatepeptide. See FIGS. 2 and 3. The data indicate that mice immunized withBK108 (with CpG DNA, PADRE and incomplete Freund's adjuvant) respondedby producing cytotoxic T lymphocytes that specifically recognize thisBKV VP1 epitope.

Example 3 In Vivo Processing of VP1 Generates the Peptide Epitope, BK108

A transgenic murine model was used to test whether the peptides thatshowed positive HLA-A2 binding in vitro were generated by in vivocellular processing and displayed on the surface of antigen-presentingcells. A recombinant Modified Ankara Virus (MVA) was constructed toexpress the BKV VP1 polypeptide as follows: nucleotide sequencescorresponding to the BKV antigen and VP1 open reading frame sequences ofinterest were cloned by PCR amplification using pBKV (33-1; ATCC #45024)as a template. The amplification products were cloned into pCR2.1, andverified by nucleotide sequencing before re-cloning into therecombination vector pLW22-1. Generation and selection of rMVAs wasperformed using previously described methods. See Wang et al., J. Virol.78:3965-3976, 2004. Verification of expression was done byimmunostaining of BHK monolayers infected with the rMVAs using ananti-polyomavirus antibody (Novocastra™).

The MVA recombinant expressing the VP1 polypeptide was used to immunizetransgenic HHD-II mice (knockout for murine class I alleles andexpressing a chimeric HLA-A2 and Kb molecule) with no adjuvant orbooster immunizations. HHDII mice (8-12 weeks old) were immunizedintraperitoneally with 3 to 5×10⁷ pfu of rMVA expressing BKV VP1.Expression of the BKV polypeptide was verified by immunostaining ofinfected BHK monolayers. The animals were sacrificed after 2 weeks andthe spleens retrieved. Single-cell splenocyte suspensions were preparedby passing the cells through a 70 μm cell strainer using the plungerfrom a sterile 1 mL syringe. Parallel in vitro stimulation cultures wereset up using each of the four peptides BK26, BK27b, BK108 and BK107.Splenocytes were subjected to one round of in vitro expansion accordingto the methods of La Rosa et al., Blood 100:3681-3689, 2002. Briefly,the splenocytes from immunized animals were co-cultured withpeptide-loaded lipo-polysaccharide (LPS) blasts in complete IVS medium(RPMI medium supplemented with 10% FCS, with glutamine, penicillin andstreptomycin) at a ratio of 3:1 for 7 days, with the addition of 10% ratT-stim™. At the end of this in vitro expansion step, the splenocyteswere tested for their ability to lyse naïve syngeneic splenocytes pulsedwith the cognate peptides (cytotoxicity) and for their ability toproduce IFN-γ on stimulation with these same peptides in ICC assays.

Cytolytic activity of effector cell populations was determined using a4-hour chromium release assay (CRA) following one in vitro stimulationaccording to the methods of Daftarian et al., J. Immunol. 171:4028-4039,2003 and La Rosa et al., Blood 97:1776-1786, 2001. The target cells wereJurkat A2.1 cells pulsed with 10 μM of the relevant or control HIVpeptides or infected for 2-3 hours with 15 MOI of rMVA expressing BKV Tantigen, VP1 or control polypeptides. For the assays, the Jurkat A2.1target cells were loaded with 200 μCi of Na⁵¹ CrO₄ ⁻ for 1 hour in a 37°C. water bath and further processed as described in La Rosa et al.,Blood 97:1776-1786, 2001, the disclosures of which are herebyincorporated by reference. Experimental evaluations were performed intriplicate.

For ICC assays, splenocytes, after 1 week in vitro stimulation, weretested for IFN-γ production after stimulation overnight with 5 μM BK108peptide or control. The following day, brefeldin A was added to all thecultures and incubation continued for 4 hours. The cells then werewashed with 3 mL PBS containing 0.5% BSA before labeling for 20 minutesat 4° C. with FITC-conjugated murine CD8-specific antibody. The cellsthen were washed again with PBS containing 0.5% BSA, permeabilized withCytofix/Cytoperm™ and labeled with APC- or PE-conjugated anti-IFN-γantibody 30 minutes at 4° C. The cells then were washed and analyzed byflow cytometry.

Results of the cytotoxicity and ICC assays are presented in FIGS. 4 and5. FIG. 4 shows the percent cytotoxicity of targets loaded with twodifferent preparations of BK108 peptide (diamonds and triangles) or withan irrelevant control peptide (squares) by splenocytes immunized in vivowith BK108. FIG. 5 provides data from ICC assays performed on cells withcontrol HIV peptide (FIG. 5A) and with BK108 peptide (FIG. 5B). Asignificantly larger proportion of BK108-stimulated cells (3.89%)responded compared to control cells stimulated with an irrelevant HIVpeptide (0.35%). Cells responding to stimulation with IFN-γ productionare circled in FIG. 5B. Of the tested BKV peptides, only peptides BK108(LLMWEAVTV; SEQ ID NO:1) and BK107 (NLLMWEAVTV; SEQ ID NO:2) yieldedpositive results. Therefore, in the immunized mice, VP1 polypeptide,expressed by the MVA recombinant, was processed within mouse cells, invivo, (1) to yield the antigenic epitope corresponding to peptide BK108,presented on the surface of antigen presenting cells and (2) to inducethe generation of CTL recognizing this same epitope.

The JCV homolog (JC100; ILMWEAVTL; SEQ ID NO:3), which differs at the Cterminal and N terminal positions from the BKV VP1 sequence, has beendescribed as a functional HLA-A*02-restricted cellular epitope inhumans. See Du Pasquier et al., J. Virol. 77:11918-11926, 2003; DuPasquier et al., J. Virol. 78:10206-10210, 2004; Koralnik et al., J.Immunol. 168:499-504, 2002. The above experiments were repeated usingBK108-immunized splenocytes assayed for their ability to recognizetarget cells presenting this JCV homolog sequence.

The peptide, JC100, was synthesized and compared with the BK108 peptidein cytotoxicity and ICC assays using the murine effectors elicited byimmunization with rMVA-BKV VP1 and expanded by stimulation with BK108,as described above. Transgenic HHD-II mice (4 per group) were immunizedintraperitoneally with 3×10⁷ plaque forming units of rMVA-BKV VP1. Twoweeks after immunization, the mice were sacrificed, spleens harvested,and the splenocytes cocultivated with syngeneic irradiatedpeptide-pulsed naïve mouse splenocytes. After 1 week of in vitrostimulation, the cultured cells were tested for specific cytotoxicityversus A2-Jurkat cells pulsed with peptides and in ICC assays for IFN-γproduction on peptide stimulation. Results show that the transgenicmurine CTL elicited by immunization with the BKV epitope recognized boththe BKV and JCV VP1 homologs, with only a somewhat higher affinity forthe BKV sequence. See FIGS. 6-8.

FIG. 6 shows killing by BK108-immune splenocytes of targets presentingBK108 (diamonds), JC100 (squares) or control HIV peptide (triangles). Intwo separate experiments, 31.01% (FIG. 7A) or 38.4% (FIG. 8B) of thecells produced IFN-γ in response to BK108, while 17.29% (FIG. 7B) or22.2% (FIG. 8C) of the cells produced IFN-γ in response to JC100,compared to 1.94% (FIG. 7C) or 3.2% (FIG. 8A) for the control peptide.Thus, protective immune responses against the BKV epitope can protectagainst JCV. In addition, this peptide should protect against SV40,which has an epitope previously identified as ILMWEAVTV (SEQ ID NO:4),because this sequence differs by only one amino acid from each of theBKV and JCV sequences (see Table I). Since most adult humans areinfected with both JCV and BKV, and SV40 also is highly prevalent, thismultifactorial protection can be important both for prophylacticvaccination and for treatment, particularly in immunosuppressedpatients.

Example 4 Identification of BKV VP1 Epitope-Specific CD8⁺ T Cells inHumans

To confirm that the cross-reactivity between BKV and JCV VP1 epitopesseen in the transgenic mouse model also exists in humans, a HLA-A*0201tetramer reagent incorporating the BK108 epitope peptide was preparedand conjugated to the fluorochrome allophycocyanin (APC) using knownmethods. See Krausa et al., Tissue Antigens 47:237-244, 1996, thedisclosures of which are hereby incorporated by reference. This tetramerreagent (termed BK108tet-APC) was used to screen PBMC samples from tenrandomly-selected healthy donors and two kidney transplant recipients,each expressing the HLA-A*0201 phenotype.

HLA typing for all donors was performed by PCR according to knownmethods. See Krausa and Browning, Tissue Antigens 47:237-244, 1996. PBMCfrom ten healthy normal HLA-A*02 haplotype donors and from two kidneytransplant recipients (designated KTx#04 and KTx#07) were collected.KTx#04 had documented BKV viremia and viruria, but no biopsy evidence ofBKVN; KTx#07 had documented viruria, but no viremia or biopsy evidenceof BKVN. The PBMC were stimulated once in culture with BK108 in thepresence of 30 U/mL rIL2 (added at day three). After a 14-day incubationperiod, the cultures were stained with FITC-conjugated antibody to CD8and with BK108tet-APC reagent for flow cytometry analysis according tothe methods of Lacey et al. Transplantation 74:722-732, 2002. Resultswere obtained using gates set on lymphocytes by forward versus sidescatter.

The cytometry results are shown in FIG. 9A-9L. See also the key in TableIII, below. The data indicated that after this in vitro expansion, PBMCfrom two of the ten healthy donors had obvious populations of CD8⁺ Tlymphocytes that bound the BK108 tetramer reagent (see 9D and 9G).Tetramer reagent-binding populations are circled for emphasis, and theirfrequencies indicated as a percentage of CD8⁺ T-lymphocytes. Two moreindividuals also may have had very small tetramer-binding populationsthat were difficult to resolve from the assay background (see, forexample, 9B). Labeling of unstimulated PBMC from the positive healthydonors with BK108tet-APC did not detect any specific binding abovebackground, indicating that the levels of CTL precursors in PBMC isbelow the detection limit of the tetramer-binding assay (approximately0.05% of CD8⁺ PBMC).

One of the two kidney transplant recipients tested harbored a very largepopulation of BK108tet-APC binding cells after stimulation (see FIG.9L). The frequency of these cells was ten-fold higher than seen in thetwo positive normal donors (24.9% of CD8⁺ T cells after amplificationversus 1.9% and 3.3%). These findings confirm the relevance of thefindings in the well-accepted transgenic mouse model to humans.

TABLE III Key to FIG. 9. Figure Donor 9A normal 1 9B normal 2 9C normal3 9D normal 4 9E normal 5 9F normal 6 9G normal 7 9H normal 8 9I normal9 9J normal 10 9K KTx#04 9L KTx#07

Example 5 Functionality and Specificity of BK108 Tetramer-Binding CD8⁺ TCells

A modified flow-based assay confirmed that CD8⁺ T cells bound by theBK108 tetramer reagent were functional CTL that specifically recognizedthis epitope. This assay combined tetramer reagent staining,intracellular cytokine staining and measurement of mobilization ofcytotoxic granules. See Betts et al., J. Immunol. Meth. 281:65-78, 2003;Betts et al., J. Immunol. 172:6407-6417, 2004; Wolint et al., J. Exp.Med. 199:925-936, 2004; Lacey et al., J. Infect. Dis. 191:977-984, 2005for relevant methods. The disclosures of these references are herebyincorporated by reference.

In this type of assay, PBMC first are stained with a tetramer reagentfor the antigenic peptide of interest, then stimulated for 4 hours inculture with the same peptide in the presence of co-stimulatoryantibodies and fluorochrome labeled antibodies specific for thelysosome-associated membrane proteins LAMP-1 (CD107a) and LAMP-2(CD107b), molecules that are present on the membranes of cytotoxicgranules in these cells. Monensin also is added to the culture toinhibit secretion of cytokines and to neutralize the pH within thecytotoxic granules, avoiding quenching fluorescence of the fluorochromeconjugated to the anti-CD107a and anti-CD107b antibodies.

If the cells under study recognize the peptide, then during theincubation step engagement of the TCR by the peptide presented on theMHC—I complex of antigen presenting cells within the PBMC populationcauses (1) production of IFN-γ and (2) mobilization of the cytotoxicgranules to the cell surface where they fuse with the plasma membrane.Cytotoxic granule fusion exposes the CD107a and CD107b markers to theexterior milieu, where they are labeled by the fluorochrome conjugatedanti-CD107a and anti-CD107b antibodies. The cells are fixed,permeabilized, and stained with labeled antibodies specific for IFN-γand CD8 for flow analysis. For additional discussion of the technicalaspects of this flow-based functional assay see Betts et al., J.Immunol. Meth. 281:65-78, 2003, the disclosures of which are herebyincorporated by reference.

The combined ICC and CD107 mobilization/degranulation assays performedhere essentially followed the methods described in Betts et al., J.Immunol. Meth. 281:65-78, 2003. Cells from BK108 in vitro stimulationcultures were washed once with RPMI 10 medium. Aliquots of 1 millioncells were labeled with BK108 tetramer reagent in 100 μL of the samemedium for 30 minutes at 37° C. One milliliter of RPMI 10 medium andFITC-conjugated antibodies specifically binding CD107a and CD107b(Pharmingen™) then were added to each aliquot, followed by 1 μg/mL eachof co-stimulatory antibodies to CD28 and CD49d (Pharmingen™). Antigenicpeptide corresponding to that incorporated in the tetramer reagent orirrelevant control peptides then were added to some of the tubes to afinal concentration of 5 μM. Monensin (1 μL, GolgiStop™) was added toall the tubes, which then were incubated at 37° C. in a CO₂-gassedincubator for 5 hours. The cells then were washed with 3 mL PBScontaining 0.5% BSA and labeled for 20 minutes at 4° C. withPerCP-conjugated antibody to CD8 (Pharmingen™). The cells then werewashed again with PBS containing 0.5% BSA, permeabilized withCytofix/Cytoperm™ and labeled with APC-conjugated antibody to IFN-γ for30 minutes at 4° C.

The cells were washed a final time and resuspended in 0.5 mL sheathfluid (FACSFlow™, Becton Dickinson™) for flow analysis. A primary gatewas set on lymphocytes using forward and side scatter, and a secondarygate was set on CD8⁺ tetramer reagent-binding cells. At least 100,000events were collected per sample. The percentage of CD8⁺ tetramerreagent-binding lymphocytes expressing elevated surface CD107a/b andsecreting IFN-γ was determined by reference to controls incubated withco-stimulatory antibodies to CD28 and CD49d but no peptides. In thisassay, the expanded cell culture was labeled with BK108tet-APC andrestimulated in culture for 4 hours with peptide in the presence ofco-stimulatory antibodies, monensin and FITC-conjugated antibodiesspecific for CD107a and CD107b. After permeabilization and labeling withCyChrome™-conjugated antibody to CD8 and PE-conjugated antibody toIFN-γ, the cells were subjected to flow analysis.

For flow analysis, a primary gate was set on lymphocytes by forwardversus side scatter and in the case of the four CD107 versus IFN-γplots, a primary gate was set on tetramer reagent-positive cells.Results of a representative assay are shown in FIGS. 10 and 11 (PBMCfrom normal donor #07 that had been stimulated for 2 weeks in culturewith BK108 peptide in the presence of 30 U/mL rIL2). FIG. 10 is aBK108tet-APC versus CD8 plot; FIG. 11 is a quartet of cD107 versus IFN-γplots (11A: no peptide; 11B: BK108; 11C: JC100; 11D: both BK108 andJC100). Similar results also were obtained in a repeated assay with PBMCfrom normal donor #04. The values in the plot quadrants indicate cellnumbers as a percentage of CD8-positive/BK108 tetramer reagent-positivelymphocytes.

Stimulation in culture with either BK108 or JC100 induced IFN-γsecretion and degranulation by 58%-59% of the BK108 tetramerreagent-binding CD8⁺ T-cells. There was a noticeable background(approximately 11%) of tetramer-reagent-binding cells with non-specificdegranulation and a smaller proportion of cells (approximately 4.5%)that displayed both degranulation and IFN-γ production. This backgroundmay have been due to the stimulation of the cells under study since thisbackground level generally is not seen with unstimulated PBMC.

Stimulation with a combination of the two peptides only slightlyincreased the proportion of cells that degranulated and produced IFN-γ,indicating a high degree of overlap between the cells responding to theBK108 peptide and those responding to the JC100 peptide. This indicatesthat the majority of the CD8⁺ T cells bound by the BK108 tetramerreagent are functional CTL that recognize both the JCV and BKV homologsof this antigenic epitope with comparable efficiency.

Example 6 Antigenic Specificity of CD8⁺ T-Cells

To definitively confirm that individual CD8⁺ T cells expanded from humanPBMC populations in response to stimulation with BK108 cross-recognizethe JC100 epitope, the cells were co-stained with a JC100 peptide HLA-A2tetramer reagent labeled with phycoerythrin (JC100tet-PE) andBK108tet-APC and subjected to flow cytometry. This assay distinguishedamong cells binding either or both of the two tetramer reagents.

Aliquots of PBMC from kidney transplant patient KTx#02 were stimulatedin culture with BK108 or with JC100 for two weeks in the presence ofIL-2 to expand cells specific for those antigens. Following theexpansion, the cultured cells were labeled with BK108tet-APC,JC100tet-PE or both. For purposes of comparison, unstimulated PBMC fromthis subject also were labeled in the same manner. The results arepresented in FIGS. 12 and 13. FIGS. 12A, 12D and 13A present data fromanalyses performed on unstimulated, uncultured cryopreserved PBMC fromkidney transplant patient Ktx#02.

FIGS. 12B, 12E and 13B present data from analyses of an aliquot of thesame PBMC, but which have been stimulated in culture with BK108 peptidein the presence of rIL2. FIGS. 12C, 12F and 13C represent analyses on analiquot of these PBMC stimulated in culture with JC100 peptide in thepresence of rIL2. The cells were labeled with BK108tet-APC (FIGS. 12A,12B and 12C), JC100tet-PE (FIGS. 12D, 12E and 12F) or both (FIGS. 13A,13B and 13C). The values shown in the plot quadrants indicatetetramer-binding cell numbers as percentages of CD8⁺ lymphocytes. Themajority of labeled cells in the two in vitro-stimulated cultures boundboth the JCV and the BKV tetramer reagents. In the absence of in vitrostimulation, labeled cells were very infrequent and difficult todistinguish from background, however staining with both labeled tetramerreagents was able to resolve these cells. The frequency ofdouble-labeled cells in the unstimulated PBMC population wasapproximately 0.6% of CD8⁺ lymphocytes in this sample. The doubletetramer-positive population within the unstimulated PBMC is circled foremphasis (FIG. 13A).

In vitro stimulation using BK108 (FIGS. 12D, 12E and 12F) expanded asizeable population of cells that bound tetramer reagent (30.3% bindingthe BKV tetramer reagent and 20.1% binding the JVC tetramer reagent).The majority (84%) of the cells bound both the BKV and JVC tetramerreagents (see FIG. 13B). Stimulation with JC100 expanded a smallerpopulation of tetramer reagent binding cells (4.4% binding the BKVtetramer reagent and 3.7% binding the JVC tetramer reagent; see FIGS.12C and 12F). This difference in expansion efficiency could reflect ahigher affinity of the CTL precursors within the PBMC for the BKVsequence.

A somewhat smaller majority (63%) of tetramer reagent binding cellsbound both tetramer reagents (FIG. 13C). This difference could reflectJCV peptide-stimulated expansion of a population of cells that bind theJCV tetramer reagent but not the BKV tetramer reagent. Therefore, theeffect of varying the relative amounts of the two tetramer reagents usedto label these samples was tested using competitive titration.Increasing the amount of JC100 tetramer reagent while keeping the amountof corresponding BKV reagent constant did not alter the proportion ofthe CD8⁺ cells binding either or both tetramer reagent. Thus, the highdegree of cross-reactivity between the BKV and JCV variants of thiscellular epitope was confirmed.

Example 7 In Vivo Processing of VP1 Generates the BK44 Peptide Epitope

JC36 (SITEVECFL; SEQ ID NO:6), which differs at the C terminal positionfrom the corresponding BKV VP1 sequence, BK44 (AITEVECFL; SEQ ID NO:5),has been described as a functional HLA-A*02-restricted cellular epitopein humans. See Du Pasquier et al., J. Virol. 77:11918-11926, 2003; DuPasquier et al., J. Virol. 78:10206-10210, 2004; Koralnik et al., J.Immunol. 168:499-504, 2002.

A well-known transgenic mouse model was used to test whether the BK44peptide was generated by in vivo cellular processing of BKV VP1 anddisplayed on the surface of antigen-presenting cells. Transgenic HHD-IImice (4 per group) were immunized intraperitoneally with 3×10⁷ plaqueforming units of rMVA-BKV VP1. Two weeks after immunization, the micewere sacrificed, spleens harvested, and the splenocytes co-cultivatedwith syngeneic irradiated BK44 peptide-pulsed naïve mouse splenocytes.After one week of in vitro stimulation, the cultured cells were testedfor specific cytotoxicity versus A2-Jurkat cells (pulsed with BK44 orJC36 peptide) and in ICC assays for IFN-γ production on peptidestimulation. Results show that the transgenic murine CTL elicited byimmunization with the BKV epitope peptide recognized both the BKV andJCV homologs, with comparable affinity. See FIGS. 14-18.

FIG. 14 shows killing by BK44-immune splenocytes of targets presentingBK44 (diamonds), JC36 (squares) or control HIV peptide (triangles). FIG.15 provides results from intracellular cytokine assays performed on thesame cells. Stimulation with BK44 peptide induced the secretion of IFN-γby CD8⁺ cells. Thus, protective immune responses against the BK44peptide epitope can protect against JCV by cross-recognition of the JC36epitope. In addition, this peptide should protect against SV40, whichhas a previously identified epitope, SV44 (SFTEVECFL; SEQ ID NO:7),because this sequence differs by only one amino acid from the JCVsequence and two amino acids from the BKV sequence (see Table I).

FIGS. 16 and 17 show representative data from analyses of an aliquot ofPBMC from a normal donor (Subject 4) which have been stimulated inculture with BK44 peptide in the presence of rIL2. The cells werelabeled with BK44tet-APC (FIG. 16A), JC36tet-PE (FIG. 16B) or both (FIG.17). When obtaining the data shown in FIG. 17, the flow cytometer wasgated on CD8⁺ lymphocytes. The majority of labeled cells in the invitro-stimulated cultures bound both the JVC and the BKV tetramerreagents, as shown by all the tetramer reagent-positive cells in theplot of FIG. 17 being in the upper right quadrant.

FIG. 18 summarizes the data from analyses of the type illustrated inFIGS. 16 and 17 on PBMC from 8 healthy normal HLA-A*02 donors. Subjects3, 4 and 7 harbored populations of CTL that could be expanded onstimulation with the BK44 peptide and that were labeled with both theBK44tet-APC and JC36tet-PE tetramer reagents. In addition, subjects 5and 6 may have had weaker responses to these epitopes. The data indicatethat CTL responses to the BK44 and/or JC36 epitopes are frequent inhealthy adults expressing the HLA-A*02 allele.

Example 8 Vaccination Against Polyomavirus

A therapeutically active (immune system modifying) antigenic peptideBK108 (SEQ ID NO:1), BK107 (SEQ ID NO:2) or BK44 (SEQ ID NO:5),according to the present invention, is administered to animmunosuppressed patient or other person in need of polyomavirusimmunity modification, either polyomavirus-seropositive orpolyomavirus-seronegative. The patient can be, for example, a kidneytransplant recipient patient or donor, or other solid organ or stem celltransplant recipient or donor, or an AIDS or cancer patient. Theadministration is given in single or multiple doses separated by a givennumber of days or weeks.

The therapeutically active antigenic peptides can be formulatedaccording to any known manner which is judged to be advantageous for theparticular individual by a person of skill. For example, each or both ofthe peptides may be administered (1) as a vaccine peptide, lipidated orunlipidated, optionally in combination with a helper peptide and/or anadjuvant), (2) as a DNA or other nucleic acid (or a vector containingsuch a nucleic acid) that expresses the peptide, optionally including anadjuvant, either separate or fused to the nucleic acid, (3) in the formof antigen presenting cells that present the peptide on their surface,for example T cells or dendritic cells, or (4) in the form of artificialantigen presenting cells such as described in Oelke et al., Nat. Med.9:619-625, 2003, the disclosures of which are hereby incorporated byreference.

For peptide formulations, the patient preferably is administered about0.5-20 mg naked peptide, subcutaneously or intranasally in a suitablecarrier. Preferably, the formulation also contains a CpG DNA adjuvantand a T helper peptide, and may also contain other ingredients, forexample diluents, preservatives, buffers, anesthetics, fragrances andthe like. For viral vaccines, the patient preferably is administered106-109 infectious units of an MVA viral recombinant that contains andexpresses DNA that encodes the peptide or a fusion of the peptide and aT helper peptide, under the control of a suitable promoter.Alternatively, the patient is injected intramuscularly with about 0.1-5mg endotoxin-free DNA diluted in sterile saline or any other suitablepharmaceutical carrier, according to known methods. For cellular vaccinecompositions, T cells transfected in vitro with the DNA-based vaccinediscussed above are administered intravenously.

Example 9 Immune Responses to BK108, JC108, BK44 and JC36 in NormalDonors Expressing HLA-A2

The prevalence of immune responses to the BK108, JC100, BK44 and JC36epitopes was evaluated in a panel of 30 healthy immunocompetentvolunteers expressing HLA-A2 using the methodologies described inExample 6 (expansion of PBMC by stimulation with peptide followed bytetramer labeling and flow analysis). Results are presented in FIGS.19-22 and Table IV, below. The volunteers were randomly selected withoutprior knowledge of their polyomavirus serostatus. Not all combinationsof peptide in vitro stimulation and tetramer reagent labeling wereperformed on samples from all 30 donors and double tetramer labeling wasnot performed on samples that were negative with the single tetramers.FIG. 19 shows staining results from flow cytometry using BK108 tetramerreagent. FIG. 20 shows the same data using JC100 tetramer reagent. FIGS.21 and 22 provide data from double-staining cytometry with both BK108and JC100 tetramer reagent after in vitro stimulation with BK108 orpeptide, respectively.

Table IV Polyomavirus Immune Response Survey.

N/A indicates data not available; OS indicates off study.

With the BK108 tetramer reagent, 4 of 30 and 5 of 20 individuals werepositive for staining after in vitro stimulation with BK108 and JC100peptide, respectively. For JC100 tetramer reagent, 2 of 20 and 6 of 20individuals were positive. Both the BK44 and JC36 tetramer reagentspositively stained cells from 8 of 17 individuals tested after in vitrostimulation with BK44 peptide. Therefore, for this incomplete data set,70.6% ( 12/17) individuals had mounted a response to either BK44/JC36 orBK108/JC100 epitopes. Subjects responding to both these epitopes formed17.6% ( 3/17) of the group.

Since the in vitro stimulation experiments with JC36 were not done, thefrequency of subjects with responses to either BK44/JC36 or BK108/JC100epitopes and the frequency of subjects with response to BK44/JC36 and toBK108/JC100 epitopes likely is higher than these data alone show. Thispanel of 30 individuals was assembled with no information as to theirBKV or JCV seropositivity, therefore it is clear that a very highproportion of healthy HLA-A2 individuals have immune responses to atleast one form of these two human polyomavirus epitopes.

1. A polyomavirus diagnostic reagent which comprises a peptide, whereinsaid peptide is selected from the group consisting of LLMWEAVTV (SEQ IDNO:1); NLLMWEAVTV (SEQ ID NO:2) and AITEVECFL (SEQ ID NO:5).
 2. Thepolyomavirus diagnostic reagent of claim 1, wherein said peptide isLLMWEAVTV (SEQ ID NO:1).
 3. The polyomavirus diagnostic reagent of claim1, wherein said peptide is NLLMWEAVTV (SEQ ID NO:2).
 4. The polyomavirusdiagnostic reagent of claim 1, wherein said peptide is AITEVECFL (SEQ IDNO:5).
 5. The polyomavirus diagnostic reagent of claim 1, wherein saidpolyomavirus is BK virus.
 6. The polyomavirus diagnostic reagent ofclaim 1, wherein said polyomavirus is JC virus.
 7. The polyomavirusdiagnostic reagent of claim 1, wherein said polyomavirus is SV40.
 8. Thepolyomavirus diagnostic reagent of claim 1, which is a tetramer reagent.9. The polyomavirus diagnostic reagent of claim 1 which is a multimerreagent.
 10. The polyomavirus diagnostic reagent of claim 1 which is anMHC-I-immunoglobulin dimer reagent.